The pathogenesis of alcoholic liver disease (ALD) remains uncertain despite considerable recent progress. We present a new hypothesis which builds upon the known effect of alcohol on homocysteine metabolism by linking this to the ER stress response and consequent fatty liver, necrosis, and apoptosis which we have observed in the intragastric ethanol fed mouse model. Our preliminary results indicate that feeding betaine reverses the hyperhomocysteinemia, ER stress and liver pathology in response to alcohol. Five aims are proposed which build on this hypothesis and preliminary results. First, we will complete a detailed characterization of ethanol-induced ER stress including the cellular and zonal compartmentation alcoholic mouse liver, the effect of ethanol on the susceptibility to ER stress and apoptotic signaling, and the effect of ER stress on susceptibility to TNF. Second, we will attempt to ascertain the contribution of key components of the ER stress response to alcoholic liver disease by exploiting various mice which have gene deletions of specific components of the ER stress response (e.g. SREBP-1, CHOP, caspase 12) or other signaling mechanisms for regulating SREBP (LXRot null mice). Third, we will determine the mechanism of hyperhomocysteinemia in response to ethanol by assessing substrate pool sizes, homocysteine metabolizing enzyme expression and tracer kinetics. These studies will allow us to determine that the liver is the major source ofhomocysteine and whether decreased remethylation and/or transulfuration are primarily responsible for excess homocysteine. Fourth, we will determine the role of homocysteine in ethanol-induced ER stress. We will employ several different strategies to strengthen the evidence for linkage of homocysteine and the ER stress response to ethanol. We will determine if alcoholic liver injury is potentiated by concomitantly raising homocysteine with either excess dietary methionine or guanidinoacetate or use of cystathionine 13- synthase +/- mice. In addition, we will assess the effect of SAMe feeding on homocysteine, ER stress and liver injury and determine if.TNF acts upstream ofhomocysteine by assessing homocysteine and ER stress in response to ethanol feeding in TNF-R1 null mice. Fifth, we will determine the mechanism of action ofbetaine feeding in protecting against ethanol by comparing it to taurine, dimethlysulfoniopropriate, and by determining ifbetaine protects in MAT1A null mice. In addition, we will assess the global effects ofbetaine feeding on hepatic gene expression to look for clues of alternative effects ofbetaine which might also contribute to its protective action. We anticipate that we will obtain important new knowledge which can be applied to the prevention and treatment of ALD.